Polyamine transporter selective  compounds as anti cancer agents

ABSTRACT

Several aromatic hydrocarbons di-substituted with a polyamine are described according to formulas selected from compounds 4, 7, 10, 15 and pharmaceutically acceptable salts thereof. The novel dimeric polyamines of the present invention demonstrate enhanced penetration into cells having an upregulated polyamine transport system, such as various types of cancer cells. The disclosed aromatic polyamine dimers provide highly efficient drugs for targeting cancer cells with active polyamine transporters.

RELATED APPLICATION

This application is a divisional of U.S. Ser. No. 12/113,540 filed May1, 2008 and further claims priority to provisional application Ser. No.60/915,448 filed on May 2, 2007, which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the field of cancer treatment and, moreparticularly, to polyamines which, due to their special structure,preferentially enter cells having an upregulated polyamine transportsystem.

BACKGROUND OF THE INVENTION

Polyamines are essential growth factors for cells. All cells havemethods of manufacturing polyamines from aminoacid sources. In additioncells can import polyamines from outside the cell via a process referredto as the polyamine transporter (PAT). While much is known aboutpolyamine transport in bacteria, yeast and Leishmania spp., themammalian polyamine transporter is a measureable, yet poorly describedimport process. It is an important cancer target because many cancercells are unable to produce enough polyamines to sustain their growthrate and rely on polyamine import in order to grow. Accordingly, certainpolyamines are important vectors for delivery of agents into cells viathe cells' polyamine transport system.

SUMMARY OF THE INVENTION

With the foregoing in mind, the present invention advantageouslyprovides several aromatic hydrocarbons di-substituted with a polyamine.

These compounds are useful treatment agents and gain entrance into cellsvia the cell's own polyamine transport system. In this manner, one canappend a polyamine architecture to a polycyclic aromatic architectureand facilitate its import into cells via the polyamine transporter. Inthis sense, the polyamine acts as a drug delivery ‘vector’, whichassists cellular uptake of the attached aromatic system. The presentlydisclosed hydrocarbons have formulas according to compounds 4, 7, 10,15, as shown in the figures, and include pharmaceutically acceptablesalts thereof as well as conjugates.

The invention also includes a pharmaceutically acceptable compositioncontaining at least one aromatic hydrocarbon di-substituted with apolyamine, said hydrocarbon's formula being selected from compounds 4,7, 10, 15 and pharmaceutically acceptable salts thereof.

The invention further includes a method of treating a cell, the methodcomprising contacting the cell with a compound or conjugate selectedfrom compounds 4, 7, 10, 15, combinations thereof and theirpharmaceutically acceptable salts. Preferably, in the method the cellbeing treated is a cancer cell or a cell with an increased level oftopoisomerase IIa. Where the cell being treated expresses an increasedlevel of topoisomerase IIa and the compound, conjugate, combination orpharmaceutically acceptable salt selected preferably comprises compound4.

The invention additionally includes several conjugates employingcompounds 4, 7, 10 and 15 as treatment agents which are delivered via acell's own polyamine transport system. For example, the inventionincludes an anthracene conjugate di-substituted with homospermidine,wherein said conjugate's formula comprises compound 4 orpharmaceutically acceptable salts thereof. Also included is a naphthylconjugate di-substituted with homospermidine, wherein said conjugate'sformula comprises compound 7 or pharmaceutically acceptable saltsthereof. Part of the invention is a benzyl conjugate di-substituted withhomospermidine, wherein said conjugate has a formula selected fromcompounds 10, 15, mixtures thereof and pharmaceutically acceptable saltsthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Some of the features, advantages, and benefits of the present inventionhaving been stated, others will become apparent as the descriptionproceeds when taken in conjunction with the accompanying drawings,presented for solely for exemplary purposes and not with intent to limitthe invention thereto, and in which:

FIG. 1 is a chemical flow diagram showing a synthetic scheme for makingcompounds 3 and 4 according to an embodiment of the present invention;

FIG. 2 shows a diagram of a synthetic scheme for making compounds 6 and7 according to an embodiment of the present invention;

FIG. 3 depicts a synthetic scheme for making compounds 9 and 10according to an embodiment of the present invention;

FIG. 4 presents a synthetic scheme for making compounds 14 and 15according to an embodiment of the present invention;

FIG. 5 depicts the formulas for the highly active, novel compounds ofthe present invention, compounds 4, 7, 10 and 15, compared to compoundsA, B and C, which were previously described; and

FIG. 6 shows results of several agarose gel electrophoresis analysesvisualized under UV light; these are further discussed below.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will now be described more fully hereinafter withreference to the accompanying drawings, in which preferred embodimentsof the invention are shown. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionpertains. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below.

Any publications, patent applications, patents, or other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including any definitions,will control. For example, the invention disclosed herein is anextension of the experimental work that led to application Ser. No.11/372,671, which was published as US 2007/0088081 on Apr. 19, 2007, andwhich is commonly owned with the present application and is incorporatedherein by reference in its entirety. This previously described inventionincludes parent compounds A, B and C, over which the presently disclosedcompounds represent an unexpectedly surprising and significantimprovement.

In addition, in this application the materials, methods and examplesgiven are illustrative in nature only and not intended to be limiting.Accordingly, this invention may be embodied in many different forms andshould not be construed as limited to the illustrated embodiments setforth herein. Rather, these illustrated embodiments are provided so thatthis disclosure will be thorough and complete, and will fully convey thescope of the invention to those skilled in the art. Other features andadvantages of the invention will be apparent from the following detaileddescription, and from the claims.

As used herein, the term “pharmaceutically acceptable salt” is intendedto describe any form (such as a salt of these amine systems with anorganic carboxylic acid like acetic acid or toluene-sulfonic acid ormethane sulfonic acid or an inorganic acid such as HCl, HBr, phosphoricacid, or a related group or prodrug) of a compound of the invention,which, upon administration to a subject, provides the mature or basecompound and does not cause undue harmful side-effects in the subject.Pharmaceutically acceptable salts include those derived frompharmaceutically acceptable inorganic or organic bases and acids.Suitable salts include those derived from alkali metals such aspotassium and sodium, alkaline earth metals such as calcium andmagnesium, among numerous other acids well known in the pharmaceuticalart.

For example, it is a well-known practice in pharmaceutical science touse pharmaceutically acceptable acid salts of amine derivatives tofacilitate their long storage and dosing as aqueous solutions. Theexamples listed in this invention comprise polyamine salts derived froma pharmaceutically acceptable acid (such as HCl or others) with orwithout the use of a pharmaceutically acceptable carrier (such as wateror other). Such salts can be derived from either inorganic or organicacids, including for example hydrochloric, hydrobromic, acetic, citric,fumaric, maleic, benzenesulfonic, and ascorbic acids. The pharmaceuticalcompositions obtained by the combination of the carrier and thepolyamine salt will generally be used in an effective dosage necessaryto elicit the desired biological effect. This includes its use in anantineoplastic effective amount or in a lesser amount when used incombination with other biologically active agents.

“Polyamine” relates to a polyamine compound or a polyamine conjugateaccording to an embodiment of the present invention, which have apolyamine message which is recognized by the polyamine transport systemon the surface of cells and have enhanced uptake into cells with highlyactive polyamine transporters over those which do not, (e.g., CHO vs.CHO-MG cells).

The term “transporter” is used to describe the cellular process ofbinding and/or importing a chemical entity which is outside the cell.The chemical entity in this case is the polyamine or polyamineconjugate, i.e. a polyamine compound or scaffold covalently attached toan agent such as a treatment drug or toxic agent.

The term “conjugate” is used to describe a polyamine compound accordingto the present invention which is associated with, for example,covalently bound, to a treatment agent (e.g., a cytotoxin such as ananthracenyl methyl unit) or to a known agent having anti-cancerproperties, e.g., doxorubicin.

“Cell selectivity” denotes the ability of a polyamine or polyamineconjugate of the present invention to preferentially enter in enhancedlevels cells with highly active polyamine transporters (e.g. CHO cellsor B16 melanoma cells) over those that have lower polyamine transportactivity (e.g. CHO-MG cells or Mel-A cells).

“IC₅₀ value” is the concentration of drug needed to kill 50% of therelative cell population. The lower the value the more cytotoxic thedrug is to that cell type.

“K_(i) value” reflects the affinity of the drug architecture for thepolyamine transporter. The lower the value of the K_(i), the higher theaffinity of the drug for the polyamine transporter.

L1210 cells are mouse leukemia cells and are a standard well usedbenchmark for evaluating cytotoxicity of new drug systems, especiallypolyamine containing drugs.

Chinese hamster ovary cells (CHO cells) have an active polyaminetransporter. This cell type is very susceptible to drugs which use thepolyamine transporter system to gain access to cells (i.e. polyamineconjugates).

Chinese hamster ovary cells which are chemically mutated to be polyaminetransport-deficient will be referred to as the CHO-MG cell line. Thiscell type should have lower susceptibility to polyamine conjugates whichuse the polyamine transporter to gain access to cells, since it does nothave an active transporter to facilitate their uptake.

B16 cells are melanoma, skin cancer cells with highly active polyaminetransporters. These cells should be very susceptible to polyamineconjugates which use the polyamine transporter to gain access to cells.

Mel-A cells are normal melanocytes, skin cells which have moderatelyactive polyamine transporters. These cells should be moderatelysusceptible to polyamine-conjugates, but less so than the B-16 cells.

As noted above, because cancer cells rely on polyamine transport inorder to sustain their growth rate, this provides an opportunity toselectively target rapidly dividing cancer cells via their heavyreliance on polyamine import. Drug-polyamine conjugates, which attach asmart polyamine message to a known cytotoxic agent, have been shown tohave enhanced cytotoxicity to cancer cells (mouse melanoma, B-16 cells)over their normal cell counterparts (Mel-A) in vitro. Previousanthracene-polyamine conjugates described in U.S. Pat. No. 7,001,925 aremono-substituted analogues with good selectivity for targeting the PAT.This disclosure details the synthesis and superior PAT targetingabilities of a di-substituted series of anthracene-polyamine conjugatesand other arylalkylpolyamines (di-substituted benzyl and naphthylanalogues).

The PAT targeting selectivity was determined using a Chinese hamstercell assay. The CHO wild type cells have active polyamine transportactivity and therefore should be very susceptible to the polyamineconjugate and give low IC₅₀ values. The IC₅₀ value is the concentrationof the drug required to kill 50% of the relative cell population. Thelower the value the more cytotoxic the drug is to that cell type. Incontrast, the mutant CHO-MG cells, which have no PAT activity are not assensitive to the polyamine conjugate (which is unable to enter thesecells via the defective PAT) and give higher IC₅₀ values. We expressthese as a ratio of the IC₅₀ value in CHOMG/IC₅₀ value in CHO. Thehigher the ratio the more PAT selective is the polyamine conjugate drug.Cytotoxic compounds, which do not use the PAT for cellular entry, wouldgive ratios near 1.

The previous most selective compounds were the naphthyl andanthracene-homospermidine conjugates (with CHOMG/CHO IC₅₀ ratios near150). These are described in application Ser. No. 11/372,671, which waspublished as US 2007/0088081 on Apr. 19, 2007, and which is commonlyowned with the present application and is incorporated herein byreference in its entirety, as noted above. Surprisingly, it has beendiscovered that by repeating this message (homospermidine) on the otherside of the molecule we were able to dramatically and unexpectedlyimprove the PAT targeting (e.g. CHOMG/CHO IC₅₀ ratio>2200) ability ofthese new compounds (4, 7, 10 and 15). The novel compounds of thepresent invention are synthesized via the synthetic schemes illustratedin FIGS. 1-4. In these synthetic schemes, the reagents used are asfollows: a) LiAlH₄/THF; b) PCC/CH₂Cl₂; c) 25% MeOH/CH₂Cl₂; 2-d) 50%MeOH/CH₂Cl₂/NaBH₄; e) 4N HCl/EtOH.

Synthetic Preparation of the Novel Compounds Compound 3:

(4-tert-Butoxycarbonylamino-butyl)-(4-{[10-({4-[tert-butoxycarbonyl-(4-tert-butoxycarbonylamino-butyl)-amino]-butylamino}-methyl)-anthracen-9-ylmethyl]-amino}-butyl)-carbamicacid tert-butyl ester: To a stirred solution of 2 (845 mg, 2.35 mmol) in25% MeOH/CH₂Cl₂ (20 mL), was added a solution ofAnthracene-9,10-dicarbaldehyde (250 mg, 1.07 mmol) in 25% MeOH/CH₂Cl₂(15 mL) under N2. The mixture was stirred at room temperature overnightuntil the imine formation was complete (monitored by ¹H NMR (CDCl₃)signal at 11.40 ppm disappeared). The solvent was removed in vacuo, thesolid residue dissolved in 50% MeOH/CH₂Cl₂ (40 mL) and the solution wascooled to 0° C. NaBH₄ (6.42 mmol) was added in small portions to thesolution and the mixture was stirred at rt overnight. The solvent wasremoved in vacuo, the solid residue dissolved in CH₂Cl₂ (50 mL) andwashed with 10% aq. Na₂CO₃ solution (3×30 mL). The CH₂Cl₂ layer wasseparated, dried over anhydrous Na₂SO₄, filtered and removed in vacuo togive an oily residue. The oil was purified by flash columnchromatography to yield the product 3 as a pale yellow viscous oil(92%), R_(f)=0.3 (5% MeOH/0.5% NH₄OH/CH₂Cl₂; ¹H NMR (CDCl₃) δ 8.36 (d,4H), 7.50 (d, 4H), 7.48 (m, 2H), 4.82 (br m, 2H), 4.68 (s, 4H), 3.13 (m,12H), 2.87 (t, 4H), 1.65-1.30 (m, 52H); ¹³C NMR (CDCl₃) δ 155.9, 155.4,131.9, 129.9, 125.6, 124.8, 79.1, 78.9, 53.5, 50.3, 46.9, 46.7, 46.0,40.2, 28.6, 28.5, 27.5. HRMS (FAB) m/z calcd. for C₅₂H₈₄N_(6O8)(M+H)+921.6423, found 921.6414.

Compound 4:

N-(4-Amino-butyl)-N′-(10-{[4-(4-amino-butylamino)-butylamino]-methyl}-anthracen-9-ylmethyl)-butane-1,4-diamine,Hydrochloride salt: A solution of BOC-protected 3 (890 mg, 0.97 mmole)was dissolved in absolute ethanol (13 mL) and stirred at 0° C. for 10minutes. A 4N HCl solution (22 mL) was added to the reaction mixturedropwise and stirred at 0 oC for 20 minutes and then at room temperatureovernight. The solution was concentrated in vacuo to give 4 as a yellowsolid in 95% yield. ¹H NMR (D₂O) δ 8.19 (d, 4H), 7.79 (d, 4H), 4.83 (s,4H), 3.30 (t, 4H), 3.11 (m, 12H), 1.79 (m, 16H); ¹³C NMR (D₂O): δ 129.7,127.7, 124.7, 124.0, 47.7, 47.2, 47.1, 42.8, 39.0, 24.2, 23.2, 23.1.HRMS (FAB) calcd for C₃₂H₅₂N₆.6HCl [(M+2H-6HCl)/2]⁺ 261.2199, Found261.2199.

Compound 6:

(4-tert-Butoxycarbonylamino-butyl)-(4-{[4-({4-[tert-butoxycarbonyl-(4-tert-butoxycarbonylamino-butyl)-amino]-butylamino}-methyl)-naphthalen-1-ylmethyl]-amino}-butyl)-carbamicacid tert-butyl ester: To a stirred solution of 2 (468 mg, 1.30 mmol) in25% MeOH/CH₂Cl₂ (20 mL), was added a solution ofNaphthalene-1,4-dicarbaldehyde (200 mg, 1.09 mmol) in 25% MeOH/CH₂Cl₂(15 mL) under N2. The mixture was stirred at room temperature overnightuntil the imine formation was complete (monitored by ¹H NMR (CDCl₃)signal at 11.40 ppm disappeared). The solvent was removed in vacuo, thesolid residue dissolved in 50% MeOH/CH₂Cl₂ (40 mL) and the solution wascooled to 0° C. NaBH₄ (6.53 mmol) was added in small portions to thesolution and the mixture was stirred at rt overnight. The solvent wasremoved in vacuo, the solid residue dissolved in CH₂Cl₂ (50 mL) andwashed with 10% aq. Na₂CO₃ solution (3×30 mL). The CH₂Cl₂ layer wasseparated, dried over anhydrous Na₂SO₄, filtered and removed in vacuo togive an oily residue. The oil was purified by flash columnchromatography to yield the product 6 as a pale yellow viscous oil(60%), R_(f)=0.3 (7% MeOH/0.5% NH₄OH/CH₂Cl₂; ¹H NMR (CDCl₃) δ 8.12 (m,2H), 7.50 (m, 2H), 7.38 (s, 2H), 4.87 (br m, 2H), 4.19 (s, 4H), 3.10 (m,12H), 2.73 (t, 4H), 1.65-1.35 (m, 52H); ¹³C NMR (CDCl₃) δ 155.9, 155.4,135.3, 132.0, 125.7, 125.4, 124.2, 79.1, 51.7, 49.7, 46.9, 46.7, 40.2,28.5, 28.5, 27.4, 26.7, 26.1, 25.8. HRMS (FAB) m/z calcd. forC₄₈H₈₂N_(6O8) (M+H)+871.6267, found 871.6211.

Compound 7:

N-(4-Amino-butyl)-N′-(4-{[4-(4-amino-butylamino)-butylamino]-methyl}-naphthalen-1-ylmethyl)-butane-1,4-diamine,Hydrochloride salt: A solution of BOC-protected 3 (500 mg, 0.57 mmole)was dissolved in absolute ethanol (13 mL) and stirred at 0° C. for 10minutes. A 4N HCl solution (22 mL) was added to the reaction mixturedropwise and stirred at 0 oC for 20 minutes and then at room temperatureovernight. The solution was concentrated in vacuo to give 7 as a yellowsolid in 94% yield. ¹H NMR (D₂O) δ 8.20 (m, 2H), 7.78 (m, 2H), 7.72 (s,2H), 4.83 (s, 4H), 3.28 (t, 4H), 3.11 (t, 8H), 3.04 (t, 4H), 1.85-1.73(m, 16H); ¹³C NMR (D₂O): δ 131.3, 129.3, 128.6, 128.0, 123.8, 48.2,47.3, 47.2, 47.1, 39.0, 24.2, 23.2, 23.1. HRMS (FAB) calcd forC₂₈H₅₀N₆.6HCl (M+H-6HCl)+471.4170, Found 471.4155.

Compound 9:

(4-tert-Butoxycarbonylamino-butyl)-{4-[4-({4-[tert-butoxycarbonyl-(4-tert-butoxycarbonylamino-butyl)-amino]-butylamino}-methyl)-benzylamino]-butyl}-carbamicacid tert-butyl ester: To a stirred solution of 2 (885 mg, 2.46 mmol) in25% MeOH/CH₂Cl₂ (20 mL), was added a solution ofBenzene-1,4-dicarbaldehyde (150 mg, 1.12 mmol) in 25% MeOH/CH₂Cl₂ (15mL) under N2. The mixture was stirred at room temperature overnightuntil the imine formation was complete (monitored by ¹H NMR (CDCl₃)).The solvent was removed in vacuo, the solid residue dissolved in 50%MeOH/CH₂Cl₂ (40 mL) and the solution was cooled to 0° C. NaBH₄ (6.71mmol) was added in small portions to the solution and the mixture wasstirred at rt overnight. The solvent was removed in vacuo, the solidresidue dissolved in CH₂Cl₂ (50 mL) and washed with 10% aq. Na₂CO₃solution (3×30 mL). The CH₂Cl₂ layer was separated, dried over anhydrousNa₂SO₄, filtered and removed in vacuo to give an oily residue. The oilwas purified by flash column chromatography to yield the product 9 as apale yellow viscous oil (54%), _(Rf)=0.38 (6% MeOH/0.5% NH₄OH/CH₂Cl₂; ¹HNMR (CDCl₃) δ 7.17 (m, 4H), 4.83 (br m, 2H), 3.68 (s, 4H), 3.06 (m,12H), 2.55 (m, 4H), 1.60-1.21 (m, 52H); ¹³C NMR (CDCl₃) δ 155.9, 155.4,138.8, 128.1, 126.8, 79.1, 78.8, 64.3, 53.5, 49.0, 46.9, 46.7, 40.2,28.5, 28.5, 27.5, 27.3, 26.6, 26.0, 25.6. HRMS (FAB) m/z calcd. forC₄₄H80N_(6O8) (M+H)+821.6110, found 821.6083.

Compound 10:

N-(4-Amino-butyl)-N′-(4-{[4-(4-amino-butylamino)-butylamino]-methyl}-benzyl)-butane-1,4-diamine,Hydrochloride salt: A solution of BOC-protected 9 (495 mg, 0.60 mmole)was dissolved in absolute ethanol (13 mL) and stirred at 0° C. for 10minutes. A 4N HCl solution (22 mL) was added to the reaction mixturedropwise and stirred at 0 oC for 20 minutes and then at room temperatureovernight. The solution was concentrated in vacuo to give 4 as a yellowsolid in 93% yield. ¹H NMR (D₂O) δ 7.59 (m, 4H), 4.30 (s, 4H), 3.17 (t,4H), 3.11 (t, 8H), 3.05 (t, 4H), 1.78 (m, 16H); ¹³C NMR (D₂O): δ 132.4,130.9, 64.3, 51.1, 47.4, 47.3, 47.1, 39.3, 24.5, 23.4, 23.3. HRMS (FAB)calcd for C₂₄H₄₈N₆.6HCl [(M+2H-6HCl)/2]⁺ 211.2043, Found 211.2043.

Compound 12:1,3,5-Tris(hydroxymethyl)benzene.

Trimethyl-1,3,5-benzenetricarboxylate 11 (2 g, 7.9 mmol, AcrosChemicals) in dry THF (30 mL) was added through a pressure-equalizedaddition funnel into a 250-mL flask containing LiAIH₄ (0.90 g, 23.6mmol) in dry THF (65 mL) at 0 oC under a N2 atmosphere. The mixture wasallowed to warm to room temperature and was then stirred for 4 h. Thereaction was quenched by the slow addition of a 1:1 mixture of Celiteand KHSO4. The suspension was filtered, and the Celite was washed withMeOH (100mL). The solvent was removed under reduced pressure and triol12 was obtained in 78% yield (1.05g). ¹H NMR of the product matched thatof the authentic material: ¹H NMR (300 MHz, DMSO) δ 7.18 (s, 3H), 4.50(s, 6H).

Compound 13:

1,3,5-Triformyl benzene. 1,3,5-Tris(hydroxymethyl)benzene: (1.05g, 6.25mmol) was suspended in CH₂Cl₂ (25 mL), and solid pyridiniumchlorochromate (5.98 g, 27.74 mmol) was added. After 30 min of stirring,the reaction mixture was diluted with acetone (10 mL), and was allowedto stir for 3 h. The precipitated chromium salts were filtered off andwashed with CH₂Cl₂. The organic phase was washed with a saturatedsolution of aqueous Na₂CO₃ three times, and dried over anhydrous Na₂SO₄.Filtration through a silica gel column eluting with CH₂Cl₂ afforded 13as white crystals (51%, 0.51 g). ¹H NMR (300 MHz, CDCI₃) δ 10.21 (s, 3H,CHO), 8.66 (s, 3H, aromatic).

Compound 14:

{4-[3,5-Bis-({4-[tert-butoxycarbonyl-(4-tert-butoxycarbonylamino-butyl)-amino]-butylamino}-methyl)-benzylamino]-butyl}-(4-tert-butoxycarbonylamino-butyl)-carbamicacid tert-butyl ester. 1,3,5-Triformyl benzene (0.180 g, 1.11 mmol) wasdissolved in 25% MeOH/CH₂Cl₂ (10 mL). A solution of Boc-protectedhomospermidine 2 (1.44 g, 4.011 mmol) in 25% MeOH/CH₂Cl₂ (10 mL) wasadded via an addition funnel. The reaction mixture was stirred overnightunder a N2 atmosphere. Loss of the starting material was monitored via¹H NMR spectroscopy and the disappearance of the aldehyde proton at10.21 ppm. Upon conversion of the starting material, the solvent wasextracted in vacuo and the crude material was redissolved in a solutionof 50% MeOH/CH₂Cl₂. To this new solution was added NaBH₄ (0.45 g, 11.9mmol) at 0 oC. The solution was stirred overnight under a N2 atmosphere.The solvent was removed in vacuo and flash column chromatography (1%NH₄OH/5% CH₃OH/CH₂Cl₂) provided a mixture of the co-elutingBoc-protected homospermidine 2 and the desired product 14 (1.08 g). Toaid in the chromatographic separation of 14 and 2, another reaction wascarried out. The mixture was dissolved in THF (45 mL) and stirred for 20min at 0 oC. A solution of2-(tert-butoxycarbonyloyimino)-2-phenylacetonitrile) (BOC-ON, 0.23 g,0.93 mmol) was added dropwise with constant stirring. After the additionwas complete, the reaction was stirred for 2 h at 0 oC under a N2atmosphere. Upon completion, the solution was concentrated in vacuo, theresidue was redissolved in CH₂Cl₂, and washed with a saturated aqueousNa₂CO₃. The organic layer was separated, dried over anhydrous Na₂SO₄,filtered and concentrated. Flash column chromatography of the residuegave pure 14 as colorless oil (79 mg). Yield: 6%. _(Rf)=0.35 (1%NH₄OH/6.5% CH₃OH/CH₂Cl₂); ¹H NMR (300 MHz, CDCl₃) δ 7.14 (s, 3H,aromatic), 4.69 (s, 3H, NH), 3.75 (s, 6H, CH2), 3.15 (m, 18H, CH2), 2.65(t, 6H, CH2), 1.65-1.15 (m, 78H, CH2, CH3); ¹³C NMR (CDCl₃): δ 156.0,155.6, 140.5, 126.7, 79.3, 79.2, 54.1, 49.5, 47.1, 46.9, 40.4, 28.7,28.6, 27.6, 27.5, 26.8, 26.1, 25.8. HRMS (FAB) m/z calcd forC63H117O12N9 (M+H)⁺ 1192.8894, found 1192.9008.

Compound 15:

N-(4-Amino-butyl)-N-(3,5-bis-{[4-(4-amino-butylamino)-butylamino]-methyl}-benzyl)-butane-1,4-diamine,nona-Hydrochloride Salt. Compound 14 (79 mg, 0.066 mmol) was dissolvedin absolute ethanol (8 mL) and stirred at 0° C. for 10 minutes. 4N HClsolution (10 mL) was added dropwise to the reaction mixture and stirredat 0 oC for 20 minutes and then at room temperature overnight. Thesolution was concentrated in vacuo to give 15 as a white solid in 97%yield (59 mg). ¹H NMR (300 MHz, D₂O) δ 7.64 (s, 3H, aromatic), 4.32 (s,6H, CH2), 3.18 (t, 6H, CH2), 3.07 (m, 18H, CH2), 1.77 (m, 24H, CH2); ¹³CNMR (D₂O): δ 135.6, 135.1, 53.2, 49.8, 49.7, 41.7, 26.9, 25.8, 25.7;HRMS (FAB) m/z calcd for C₃₃H₇₈N₉Cl₉ (M+H-9HCl)⁺ 592.5749, found592.5749.

TABLE 1 Elemental analyses for compounds Compound Molecular C H N No.Formula Calcd. Found Calcd. Found Calcd. Found 3 C₅₂H₈₄N₆O₈•0.4H₂O 67.2767.23 9.21 9.07 9.05 8.87 4 C₃₂H₅₈N₆Cl₆•1.3H₂O 50.38 50.36 8.01 8.0511.02 10.84 6 C₄₈H₈₂N₆O₈ 66.18 65.99 9.49 9.47 9.65 9.57 7C₂₈H₅₆N₆Cl₆•2.5H₂O 45.79 45.94 8.37 8.25 11.44 11.37 9 C₄₄H₈₀N₆O₈•0.2H₂O64.07 64.03 9.83 9.84 10.19 9.94 10 C₂₄H₅₄N₆Cl₆ 45.08 44.85 8.51 8.4313.14 12.86 14 C₆₃H₁₁₇O₁₂N₉•0.5H₂O 62.97 9.90 10.49 62.85 9.90 10.39 15C₃₃H₇₈N₉Cl₉•1.5H₂O 41.85 8.62 13.31 41.90 8.53 13.18

TABLE 2 CHO studies CHOMG/CHO Compound CHOMG IC₅₀ (μM) CHO IC₅₀ (μM)IC₅₀ Ratio A 66.7 (±4.1) 0.45 ± 0.10 148 B >100 0.6 ± 0.2 >164C >1000  >1000 NA  4 >100 0.045 ± 0.003 >2222  7 >100 0.12 ± 0.03 >83310 50.2 ± 3.8 0.074 ± 0.010 677 15 >500  >500 NA

TABLE 3 L1210 studies L1210 + DFMO K_(i) (μM) L1210 IC₅₀ (μM) IC₅₀ (μM)A 1.8 ± 0.1 0.30 ± 0.04 0.09 ± 0.01 B 3.8 ± 0.5 0.50 ± 0.03 0.43 ± 0.02C 4.5 ± 0.8 36.3 ± 8.4  ND  4 0.39 ± 0.05 0.78 ± 0.07 0.22 ± 0.08  70.17 ± 0.02 0.25 ± 0.08 ND 10 0.52 ± 0.11 0.16 ± 0.01 0.36 ± 0.03 150.49 ± 0.02 122.3 ± 8.1  49.3 ± 10.0 ND = not determined

Biological Evaluation of the Compounds

The biological evaluation of these compounds is shown in Table 2. Asindicated therein, compound 4 was found superior in terms of targetingCHO cells with active polyamine transport showing a surprising IC₅₀ratio of >2200! This is an order of magnitude higher than found with ourpreviously reported compounds A and B. In our earlier report, compound Cwas shown to be somewhat unstable to cellular oxidases and partiallydegraded into homospermidine. As such, we were unable to measure itscytotoxicity and C had an IC₅₀ value >1000 μM in both CHO and CHOMG celllines. Cytotoxicity experiments were also performed in murine leukemiacells (L1210, Table 3) with and without the presence ofdifluoromethylornithine (DFMO), a known inhibitor of ornithinedecarboxylase (the enzyme responsible for polyamine biosynthesis).Typically, when one blocks the ability of cells to synthesizepolyamines, the cells respond by increasing their import ofextracellular polyamines via PAT. Therefore, if our drugs arePAT-selective we should see a lowering of their respective IC₅₀ valuesin the presence of DFMO, a molecule which facilitates the drug's import.Indeed, as shown in Table 3, the IC₅₀'s are lower in the presence ofDFMO, except for 10. K_(i) values indicate the binding affinity of thecompounds for the PAT. Using competition experiments with radiolabeledspermidine, we were able to rank the binding affinity of the compoundsfor the PAT. Low K_(i) values mean the compound has a high bindingaffinity for the PAT. As shown in Table 3, the di-substituted analogues4, 7, and 10 all had significantly lower K_(i) values (vs. A-C,respectively) and were better PAT binding agents.

Compound 4 as a Topoisomerase II (Topo-II) Catalytic Inhibitor:

Topoisomerase IIalpha (Topo IIa) is known to be highly expressed inrapidly multiplying cells such as cancers. In sheath tumors of theperipheral nerves, a difficult to treat cancer, high expressed levels ofTopo Ila are associated with poor prognosis and survival.

Previously, we have tested several branched polyamine-anthracene andacridine conjugates for their ability to inhibit Topo IIa and theresults were shown to be positive for all tested conjugates (seePhanstiel et al., J. Org. Chem., 2000, 65, 5590-5599; which isincorporated herein by reference in its entirety). Thus the linearpolyamine-anthracene conjugates herein disclosed (e.g., compound 4) wereevaluated as Topo II inhibitors. A well established in vitro Topo IIassay (as used previously) allowed us to determine whether these newconjugates (previously described compound A and novel compound 4 of thepresent invention) have the ability to inhibit Topo II by preventingTopo IIa from decatenating (i.e., disentangling) kinetoplast DNA (kDNA).It was concluded that conjugates A and 4 were catalytic Topo-IIinhibitors, where inhibition correlated positively with an increase indrug concentration. Compound 4 was superior to the previously describedparent compound A, where good inhibition was observed at a concentrationof 5 μM with compound 4 (i.e., significant kDNA remained undecatenatedin the top well of FIG. 6, indicating Topo II inhibition). In contrast,significantly less inhibition was observed at the same concentration (5μM) with compound A. Both conjugates were compared to a known inhibitor,Berenil (labeled ‘B’ in the last lane of each gel in FIG. 6), as acontrol.

In this regard, FIG. 6 shows inhibition of topoisomerase II. A negativeimage of agarose gel electrophoresis is visualized under UV light. Drugswere incubated with kDNA (200 ng) and Topo IIa (4 units) in reactionbuffer prior to loading onto the gel. Neg. Control (−): kDNA only; Pos.Control (+): kDNA+Topo II (without conjugate); Inhibitory Control (B,rightmost lane): Berenil (50 μM). All other lanes contain thecorresponding conjugate at the listed μM concentrations. Positive TopoII activity is shown by the formation of nicked open circular (NOC) DNA(middle band) and covalently closed circular (CCC) DNA (bottom band).Inhibition is shown by the negative activity of Topo II where kDNA (topband) is not decatenated and remains in the well.

When kDNA was not incubated with drug and Topo II (negative control), nomigration occurred on the gel and the kDNA remained in the well asexpected, which was indicated by the single top band visualized in thewell. When kDNA was incubated with Topo II in the absence of drug, thetop band in the well disappeared completely while two bands representingthe two decatenated products could be seen. This showed that the enzymehad good activity and only inhibition rather than enzyme inactivityproduced the band in the top well upon added drug. Berenil, a knowninhibitor at 50 μM showed good inhibition as expected and gave most ofthe kDNA remaining in the well.

Accordingly, compound 4 appears to be a novel inhibitor of Topo II and,as such, is a potential agent for use against cancers where increasedlevels of the enzyme play a significant role in the pathogenesis of thedisease.

In addition, novel compounds 4, 7, 10 and 15 represent the nextgeneration of smart drugs and a major advance in terms of PAT-selectiveagents. These compounds have similar cytotoxicities to the originalseries A-C in L1210 cells, but are significantly more efficient inentering cells with active polyamine transporters (e.g. CHO cells). Thisis represented by their 10-fold increase in potency in the CHO wild typecell line over their A-C counterparts. Additionally, compounds 4 and Amay also be used as Topo II catalytic inhibitors in vitro as they showedgood inhibition in a well-established assay.

Accordingly, in the drawings and specification there have been disclosedtypical preferred embodiments of the invention and although specificterms may have been employed, the terms are used in a descriptive senseonly and not for purposes of limitation. The invention has beendescribed in considerable detail with specific reference to theseillustrated embodiments. It will be apparent, however, that variousmodifications and changes can be made within the spirit and scope of theinvention as described in the foregoing specification and as defined inthe appended claims.

That which is claimed:
 1. A method of treating a cell, the methodcomprising contacting the cell with a compound or conjugate selectedfrom compounds 4, 7, 10, 15, combinations thereof and theirpharmaceutically acceptable salts.
 2. The method of claim 1, wherein thecell treated is a cancer cell.
 3. The method of claim 1, wherein thecell treated expresses an increased level of topoisomerase Ila and thecompound, conjugate, combination or pharmaceutically acceptable saltselected comprises compound
 4. 4. The method of claim 1, wherein thecell treated expresses a polyamine transport system and the compound,conjugate, combination or pharmaceutically acceptable salt selectedcomprises compound
 15. 5. The method of claim 4, wherein compound 15 isused in an effective amount to at least partially inhibit the polyaminetransport system of the cell.